| BackgroundSepsis refers to the body for the infection of the uncontrolled reaction can lead to life-threatening organ failure,is one of the important causes of death in critically ill patients.According to foreign epidemiological survey,in intensive care unit non-heart disease patients,sepsis mortality has exceeded myocardial infarction and become the main reason for death.Studies have shown that: SIRS is the body's non-specific immune system on bacterial endotoxin and its pro-inflammatory factors over-reaction.Tyrosine kinases are divided into receptor tyrosine kinases and non-receptor tyrosine kinases.Tec family in recent years abroad active cytoplasmic tyrosine protein kinase,belonging to non-receptor tyrosine kinase.BMX(bone marrow tyrosine kinase on chromosome X)is a newly discovered member of the Tec family.Recent studies have shown that BMX kinases are involved in the development of chronic inflammatory diseases and tumors such as rheumatoid arthritis and renal cell carcinoma.In this experiment,RAW264.7 mouse mononuclear macrophages were used as experimental materials.LPS induced cell inflammatory response.Intervention with BMX-specific inhibitor BMX-IN-1.To observe the effect of BMX kinase on the production of pro-inflammatory cytokines TNF-? and IL-1? induced by LPS and to explore its possible molecular mechanism to elucidate the early inflammatory mechanism of sepsis and to provide advice on its prevention and treatment.PurposeToinvestigate the role of non-receptor tyrosine kinase BMX in the production of TNF-? and IL-1? from macrophages induced by lipopolysaccharide.Material and method PART ? The effect of LPS on the expression of BMX kinase in RAW264.7 macrophagesMouse RAW264.7 mononuclear macrophages were routinely cultured after LPS dose effect and time effect of two aspects of the study.(1)Dose effect study: Different concentrations of LPS(0.01?0.1?1?10?100?g / m L)were added for 24 h.(2)Time effect study: 0.1 ?g / m L LPS was used to stimulate 0?15min?30min?45min?60min and 120 min respectively.The expression and phosphorylation of BMX kinase were detected by Western BlotPART? Study on dose and time effect of BMX-IN-1 Mouse RAW264.7 mononuclear macrophages were routinely cultured after BMX-IN-1 dose effect and time effect of BMX kinase specific inhibitor were studied in two aspects.(1)Dose effect study: The control group was cultured for 25 h.The LPS control group was cultured for 24 h,then stimulated with 0.1 ?g / m L LPS for 1 h.BMX-IN-1(75 000 nmol / L?7 500 nmol / L?750nmol / L?75nmol / L)was preincubated for 24 h,then stimulated with 0.1?g / m L LPS for 1 h.The expression of TNF-? m RNA was measured by real-time PCR.Compared with the blank control group and the LPS control group,we can get the optimum concentration of BMX-IN-1.(2)Time effect study: BMX-IN-1 was preincubated at 75 000 nmol / L for 2 h?4 h?8 h?12 h and 18 h.The expression of TNF-? m RNA was measured by real-time PCR.Compared with the LPS control group,we can get the optimum pre-treatment time of BMX-IN-1.PART?Role of non-receptor tyrosine kinase BMX in the production of TNF-? and IL-1? from macrophages induced by lipopolysaccharide and its mechanismRAW264.7 mouse mononuclear macrophages were divided into 4 groups according to the random number table method:(1)blank control group was cultured in DMEM medium for 13 hours;(2)BMX-IN-1 group(3)LPS group,traditional culture for 12 h,then stimulated by LPS for 1 h;(4)BMX-IN-1 + LPS group :BMX-IN-1 pretreated for 12 h,then stimulated by LPS for 1 h.The levels of TNF-? and IL-1? in supernatants were detected by ELISA.The expression of TNF-? and IL-1? were detected by real-time PCR.The expression of BMX and p38 MAPK were detected by Western blotting.Results PART ? The dose effect of LPSWestern Blot results showed that LPS-induced expression of BMX kinase in RAW264.7 cells was dose-dependent.LPS dose of 1 ?g / m L incubated at 24 h,RAW264.7 cells BMX kinase expression peak.When LPS concentration <1 ?g / m L,the expression of BMX kinase gradually increased with the increase of LPS concentration.When LPS concentration> 1 ?g / m L,the expression of BMX kinase gradually decreased with the increase of LPS concentration.The time effect of LPSWestern Blot results showed that LPS(0.1 ?g / m L)induced the expression of BMX kinase in RAW264.7 cells in a time-dependent manner.After incubation for 15 min,the phosphorylation level of BMX kinase in macrophages increased,reached a peak at 60 min,and then decreasedPART? The dose effect of BMX-IN-1The expression of TNF-? m RNA in each group was calculated and the blank control group was set to 1.The expression of TNF-? m RNA in macrophages was significantly higher than that in blank control group(with P values below 0.01).After pretreatment with BMX-IN-1,the expression of TNF-? m RNA in the cells decreased.The expression of TNF-? m RNA in the 75 000 nmol / L plus LPS group was 2.40 ± 0.41,which was significantly higher than that in the LPS group(with P values below 0.05).The expression of TNF-? m RNA in the 7 500 nmol / L group?750 nmol / L group and 75nmol / group was 2.65 ± 0.48?2.91 ± 0.57 and 3.00 ± 0.59,lower than the LPS group,but the difference was not statistically significant(with P values above 0.05).The time effect of BMX-IN-1The expression of TNF-? m RNA in each group was calculated,the LPS control group was set to 1.The expression of TNF-? m RNA of BMX-IN-1 pretreated 2 h,4 h,8 h,12 h,18 h group were 0.98±0.10?0.92±0.07?0.88±0.20?0.76±0.07?0.80±0.07.Compared with the LPS group,the expression of TNF-? m RNA of BMX-IN-1 pretreated 8 h,12 h,18 h group were different significantly(with P values below 0.05 or below 0.01).PART?The levels of TNF-? and IL-1? in the supernatant of blank control group were close to those of BMX-IN-1 group,The m RNA expression of TNF-? and IL-1? in cells of BMX-IN-1 group were close to those of control group(with P values above 0.05).The levels of TNF-? and IL-1? in cells of LPS group were(2293±393)pg/m L and(700±95)pg/m L.The m RNA expression of TNF-? and IL-1? in cells of LPS group were 2.97±0.17 and 3.07±0.60,respectively,which were significantly higher than those of control group(with P values below 0.01).The levels of TNF-? and IL-1? in cells of BMX-IN-1 plus LPS group were(1755±422)pg/m L and(457±135)pg/m L.The m RNA expressions of TNF-? and IL-1? of cells in BMX-IN-1 plus LPS group were 2.31±0.94 and2.55±0.73,respectively,which were lower than those of LPS group(with P values below 0.05).The activity of intracellular BMX ?TAK1and p38 MAPK of cells in BMX-IN-1 group were close to those of control group(with P values above 0.05).The activities of intracellular BMX ?TAK1 and p38 MAPK of cells in LPS group were 1.98±0.33?2.41±0.50 and 2.05±0.34,respectively,which were significantly higher than those of control group(with P values below 0.01)The activities of intracellular BMX?TAK1 and p38 MAPK of cells in BMX-IN-1 plus LPS group were 1.00±0.17?1.04±0.10 and 1.67±0.27,respectively,which were lower than those of LPS group(with P values below 0.01 or below 0.05).Conclusions1.LPS induced the expression of BMX kinase in RAW264.7 cells and was dose-dependent with LPS.The phosphorylation level of BMX kinase was time-dependent.2.The optimum concentration of BMX-IN-1 is 75 000 nmol/L and the optimum pre-treatment time is 12 hours.3.BMX increases the production of pro-inflammatory cytokines TNF-? and IL-1? from macrophages induced by LPS through TAK1-p38 MAPK signaling pathway. |
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